The invention generally relates to a control arrangement for the chromatography of liquids which compensates for the non-uniform passage of a specimen past the detector of a liquid chromatograph and, more particularly to a flow meter control arrangement capable of measuring very small flow rates to determine a valid correction factor.
Chromatographs for liquids are known, such as disclosed in German Patent (DE-PS) 32 26 398, wherein a specimen of closely related compounds is separated into its individual fractions by charging a separation column with the specimen and introducing a solvent into the separator column so that the resulting eluate, i.e., specimen and solvent, can be analyzed by successively passing each fraction of the specimen past a detector. In the case of known liquid chromatographs, to assure accurate analysis of the specimen, it is important that the individual fractions of the specimen measured by the chromatograph are each moved past the detector along at a uniform speed. Otherwise the analysis, which takes place by integrating the so-called peak areas of the signals generated by the detector, is falsified and the peak bases for each individual fraction become either too narrow or too wide. In the case of known liquid chromatographs, uniform passage of the fractions of the specimen past the detector has been accomplished by use of a removal device provided on the side of the detector that is connected to the outlet duct. This removal device ensures a discharge volume that is in each case constant for each time unit. While the above-noted known liquid chromatograph works very accurately, one particular drawback of this arrangement is that relatively high expenditures and complexity of equipment are required in order to ensure a constant flow rate.
Therefore, one object of the present invention is to provide an arrangement which compensates for the non-uniform passage of a specimen past the detector of a liquid chromatograph that is simple and eliminates the need for an expensive and complex removal control device connected to the outlet or exhaust line of the chromatograph.
In order to achieve this object, the present invention utilizes a unique approach for measuring the actual rate of flow of the eluate past the detector of the chromatograph. By means of this development, the respective deviation of the actual flow rate and of the desired flow rate can be determined and represented as a correction factor to provide an arrangement which compensates for non-uniform flow and is technically easier to implement than the previously known removal control device. When the actual flow rate and the desired flow rate correspond to one another, the correction factor is f=1. As used hereinafter, this correction factor f is defined as: ##EQU1## wherein FRi is the actual flow rate and FRs is the desired flow rate. The determination of the correction factor f is accomplished by a special flow meter which is capable of exactly determining small flow values. This flow meter can be used for determining the flow in devices other than liquid chromatographs; however, it is particularly advantageous for use in the chromatography of liquids.
One particular advantage of the present invention is that the flow meter, at any given moment during the analysis of a specimen, is capable of providing a computer or data station with the current or present valid correction factor f for processing the measured values generated from the detector. This arrangement permits the integrator of the analyzing device to operate correctly in real-time even if the actual flow rate deviates considerably from the desired flow rate. During normal use, if the work takes place in the normal manner and at a constant pressure upstream of the separation column, the deviations from the desired flow rate are not extreme. However, the individual retention times of each individual fraction of the specimen, which are used for peak identification, cannot remain uncorrected in order to assure accurate analysis of the specimen. Therefore, during the correction, the respective deviations from the desired flow rate are entered into the computation of the relative retention times and further, this process can also be fully automated by means of the appropriate hardware or software.
Another advantage of preferred embodiments of the present invention consists of using the value of the retention volume instead of the value of the retention time for peak identification. It was found that under defined conditions (column, temperature, elutriating agent, admission pressure), and also when the flow rates are fluctuating, the elutriating agent volume, which is required for moving a certain fraction through the column, fluctuates only very little. With this aforementioned approach, the flow rate can be changed without any damaging effect during an analysis, and in many cases, results in an important improvement with respect to the separation quality and the analysis time. Often, late-elutriating fractions, which require a relatively long waiting period are of special interest. By the intentional increase of the flow rate during the run, the late fractions can be elutriated off much faster. The gradients for the elutriating agent mixture and for the column temperature, which are used in methods of the conventional chromatography of liquids, are therefore joined by the flow rate gradient as a third gradient. This additional degree of freedom in the shaping of the analysis parameters opens up new possibilities for the optimizing of the chromatography of liquids.
Other objects, advantages and novel features of the present invention will become apParent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.